The overall goal of this project is to achieve long-term expression of a desired gene in vivo in a significant percentage of keratinocytes by identifying and introducing genes into keratinocyte stem cells (KSC). We are currently developing an ex vivo skin gene therapy model in which keratinocytes are engineered to express atrial natriuretic peptide or ANP in human keratinocytes as an approach to effectively treat systemic hypertension. ANP has both natriuretic or diuretic and vasorelaxant effects and generally counterbalance the effects of the renin-angiotensin-aldosterone system. Success in this hypertension model will allow us to pursue other therapeutic strategies. However, achieving long-term expression of desired genes in a significant percentage of keratinocytes has proved to be very difficult. Since unique cell surface markers for keratinocyte stem cells are not yet known, the isolation, purification, and efficient introduction of genes into these cells will be necessary for long-term expression in high percentages of keratinocytes is not yet possible. In order to achieve long-term expression, we have developed an approach using topical colchicine treatments to select for keratinocytes that express a multi-drug resistance (MDR) selectable marker gene along with a linked gene of interest, such as ANP. A significant advantage of this approach is that it does not depend on keratinocyte stem cell identification and targeting. When the selective treatment (colchicine) is applied topically, only keratinocytes that express the MDR selectable marker gene will survive and populate the epidermis. We have recently demonstrated the feasibility of this approach in an in vivo mouse model. When grafted keratinocytes that express MDR are topically selected with colchicine, long-term high-level expression of MDR is maintained in a significant percentage of cells. Quantitative PCR on the keratinocyte populations in the human skin grafts demonstrated that colchicine selected for keratinocyte stem cells expressing MDR, and prevented the gradual decline of MDR-expressing keratinocytes that occurs in the absence of colchicine. We are currently using this selectable skin gene therapy model to obtain persistent high-level gene expression and systemic delivery of the atrial natriuretic peptide or ANP gene in human keratinocytes as an approach to effectively treat systemic hypertension in a mouse model. ANP has both natriuretic or diuretic and vasorelaxant effects and generally counterbalances the effects of the renin-angiotensin-aldosterone system. We have developed bicistronic lentiviral vectors capable of expressing high levels of ANP and MDR, and these vectors have been used to transduce human keratinocytes and fibroblasts. Raft cultures or human skin equivalents have been constructed with these transduced cells and are capapble of secreting high levels of ANP. These human skin equivalents are now being grafted onto immunocompromised mice and systemic levels of human ANP will be assessed. Proof-of-concept success in this hypertension model will encourage us to pursue therapeutic strategies with other genes of interest.